Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0603250 on December 12, 2003

Published online before print December 12, 2003
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(Journal of Leukocyte Biology. 2004;75:434-442.)
© 2004 by Society for Leukocyte Biology

In situ demonstration of dendritic cell migration from rat intestine to mesenteric lymph nodes: relationships to maturation and role of chemokines

Hisashi Kobayashi*,1, Soichiro Miura{dagger}, Hiroshi Nagata*, Yoshikazu Tsuzuki{dagger}, Ryota Hokari{dagger}, Takashi Ogino*, Chikako Watanabe*, Toshifumi Azuma* and Hiromasa Ishii*

* Department of Internal Medicine, Keio University, School of Medicine, Tokyo, Japan; and
{dagger} Second Department of Internal Medicine, National Defense Medical College, Saitama, Japan

1 Correspondence: Second Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. E-mail: miura{at}me.ndmc.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DCs) are continuously transported from the intestine to mesenteric lymph nodes (MLNs). The objective of this study was to determine the migration kinetics of DCs via intestinal lymph and to investigate regulatory factors affecting their migration in vivo. DCs were obtained from spleen or thoracic duct lymph of mesenteric lymphadenectomized rats. The DCs were fluorescently labeled and injected into the subserosa of the small intestine near the cecum, and their migration patterns into MLNs were determined. Isolated DCs from intestinal lymph express intercellular adhesion molecule-1 (ICAM-1), CD11b/c, CD80/86, and major histocompatibility complex class II but maintain their ability to phagocytize latex particles, suggesting the presence of immature DCs. The isolated DCs accumulated in MLNs in a time-dependent manner with maximal accumulation at 48 h. Cytokine-induced maturation of lymph DCs did not cause a change in cell number but accelerated their transport into MLNs with a maximum at 24 h. Splenic DCs showed an intermediate level of maturation and a migration pattern similar to mature DCs. Inhibition of ICAM-1 or CD11b/c did not affect DC migration. Migration of mature DCs to MLNs was specifically blocked by desensitization of CCR7 with CCL21. In contrast, freshly isolated lymph DCs were not chemotactic for CCL21, but their migration to MLNs was mainly inhibited by desensitization of CCR6 with CCL20. The migratory ability of DCs correlates well with their degree of maturation, and different chemokine/chemokine receptor use may be the main regulator of DC migration kinetics through intestinal lymph.

Key Words: intestinal lymph • spleen • adhesion molecule • CCL20 • CCL21 • CCR6 • CCR7 • CXCL12


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DCs) are the most potent antigen-presenting cells and play a central role in the processing and presentation of antigens to T cells during the immune response. Antigens are captured by immature DCs in peripheral tissues and processed to form major histocompatibility complex (MHC)-peptide complexes. As a consequence of antigen deposition and inflammation, these DCs begin to mature, expressing molecules that will lead to binding and stimulation of T cells in the T cell areas of lymphoid tissues [1 2 3 4 ]. Specifically, upon activation, DCs travel to the lymphoid tissues such as the spleen and lymph nodes (LNs). There, DCs may complete their maturation [5 ], attract T and B cells by releasing chemokines [6 ], and maintain the viability of recirculating T lymphocytes [7 ].

In the small intestine, DCs play important roles in the initiation of mucosal immune responses. DCs reside in the subepithelial dome region of Peyer’s patches, where they capture antigens transported by overlying M cells. They are also found in the T cell-rich interfollicular region, where naïve T cells are likely activated to become effector cells [8 ]. Further, DCs are widely spread in the lamina propria of the gut and are recruited at sites of infection. DCs open the tight junction between epithelial cells, send dendrites outside of the epithelium, and sample bacteria [9 10 ]. However, even in the absence of invading pathogens, a fraction of the DC population seems to be transient. There is a continual traffic of DCs from intestinal mesenteric LNs (MLNs) via intestinal lymphatics, and under normal conditions, the traffic remains relatively constant [11 12 13 ]. The DCs that migrate in the steady state may replenish immature populations or may be on patrol to identify invaders such as other white blood cells [3 ].

How DCs know where to go is largely unknown. Lipopolysaccharides stimulate a variety of cells to produce cytokines and chemokines, including granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor {alpha} (TNF-{alpha}), interleukin (IL)-1, and macrophage inflammatory protein (MIP)-1{alpha} and -ß. These products are known to modulate DC migration and maturation [14 ]. Intradermal injection of TNF-{alpha} can increase the numbers of DCs that can be released from the draining node [15 , 16 ]. Also, the release of intestinally derived DCs into lymph is increased after the intravenous administration of endotoxin, and this release is at least partially dependent on TNF-{alpha} [17 , 18 ]. These reports suggest that the TNF-{alpha}-mediated release of DCs may be important for immune regulation in LNs draining inflammatory sites. However, there have been few previous reports examining which factors are important in the regulation of DC trafficking through intestinal lymph during uninflamed conditions.

Much of the work in this area has focused on the role of seven transmembrane-spanning, G-protein-coupled receptors, a growing family of proteins that includes receptors for a calcitonin gene-related peptide [19 ], complement C5a [20 ], and chemokines [21 , 22 ]. Very recent observations suggest that inflammatory chemokines secreted at sites of pathogen invasion control the migration of DCs that act as sentinels of the immune system. These studies have shown that immature DCs respond to many CC and CXC chemokines, which are induced by inflammatory stimuli. For example, Langerhans cells are selectively recruited by MIP-3{alpha}/CCL20 [23 ]. In contrast, CCR7 ligand secondary lymphoid-tissue chemokine/6Ckine/CCL21 and Epstein-Barr-induced 1 ligand chemokine/MIP-3ß/CCL19, chemokines specifically expressed in the T cell-rich areas of draining LNs, play a key role in the accumulation of mature DCs, which then become interdigitating DCs [24 ].

In this study, we investigated the migration kinetics of DCs as it transfers from the intestinal wall to the MLNs via intestinal lymph. We microinjected DCs into the rat intestinal wall and performed in situ observations of the MLNs. We demonstrated that DCs continuously migrate from the small intestine to MLNs under steady-state conditions but that the migration kinetics is heterogenous depending on the DC source (intestinal lymph vs. spleen) and the degree of maturation. In addition, we show that adhesion molecules on the DC surface, such as CD11b/c and intercellular adhesion molecule (ICAM)-1, are not involved in the regulation of DC migration to MLNs. Rather, several chemokine-chemokine receptor combinations are key regulators of the migration kinetics, especially CCL20/CCR6 for unstimulated lymph DCs and CCL21/CCR7 for mature DCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell isolation
Male Wistar rats (5 weeks old) were maintained on standard laboratory chow (Oriental Yeast, Tokyo, Japan). The Keio University School of Medicine Animal Research Committee (Tokyo, Japan) approved all animal procedures. For collection of lymph DCs, animals were mesenteric-lymphadenectomized and maintained for an additional 6 weeks. Mesenteric lymph samples were obtained from the thoracic duct lymph as described previously [25 ]. Fibrin was removed from lymph samples by cell strainer (Becton Dickinson Labware Europe, France), and the samples were centrifuged at 1500 g. Next, 2 x 108 cells were loaded onto 4 ml metrizamide (Sigma-Aldrich Chemical Co., St. Louis, MO), and lymph DCs were isolated by gradient density centrifugation over metrizamide [26 ].

Splenic DCs were obtained from rat spleens. Spleens were minced and digested in 2 mg/ml collagenase D (Roche Diagnostics, Meylan, France) in RPMI-1640/1% fetal calf serum (FCS) for 30 min at 37°C. EDTA (10 mM) was added during the last 5 min, and the cell suspension was then pipetted up and down several times and filtered. Cells were washed once in phosphate-buffered saline (PBS)/2 mM EDTA/1% FCS, and low-density cells were obtained after centrifugation at 780 g for 10 min on a 14.5% metrizamide gradient.

After obtaining DC-enriched fractions, DCs were selected as OX62-positive cells by magnetic cell sorting. Briefly, DCs (1x107 cells) were suspended in 80 µl PBS containing 0.5% bovine serum albumin (BSA) and 5 mM EDTA and were incubated with 20 µl anti-rat OX62-labeled magnetic cell sorter (MACS) microbeads (Miltenyi Biotech, Bergish Gladbach, Germany) for 15 min at 6–12°C. Thereafter, the DCs were passed through a separation column (type LS, Miltenyi Biotech), which was placed in the magnetic field of a MACS separator (MidiMACS, Miltenyi Biotech). The magnetically labeled OX62-positive cells are retained in the column, and after removal of the column from the magnetic field, the retained DCs were eluted as the positively selected cell fraction. These cells were washed and resuspended in ice-cold RPMI containing 5% FCS and were kept on ice until use. The purity of the DC-rich fraction was examined by immunocytochemistry for OX62, HIS24, and OX6 (all from Serotec, Kidlington, Oxford, UK) as well as appearance of micropodia as determined by Giemsa staining. Typical DCs were identified as panB-MHCII+.

Induction of DC maturation
Maturation of the DC-enriched fraction from intestinal lymph was induced by stimulation with TNF-{alpha}, IL-4, and GM-CSF [27 ]. Briefly, DCs were resuspended in culture medium supplemented with GM-CSF (20 ng/mL), IL-4 (10 ng/ml), and TNF-{alpha} (100 U/ml; all from R&D Systems, Minneapolis, MN), plated on six-well plates in 3 ml culture medium per well at a density of 5 x 105 cells/ml, and cultured overnight. The next day, the nonadherent cells were collected, resuspended in PBS with 0.1% BSA, centrifuged at 2000 rpm at 400 g for 10 min at 4°C, recovered, and further cultured in six-well plates (2x106 cells/ml) for 2 days with GM-CSF.

Analysis of phagocytosis by DCs
The phagocytic function of DCs was examined by assessing the uptake of latex microbeads [28 ]. Briefly, the DC-enriched fraction from intestinal lymph and spleen was cultured in eight-well chamber plates in RPMI 1640 containing 10% FCS and 1% antibiotics (streptomycin and penicillin). Cells (2x105) in 400 µl medium were cultured in the presence of 2.5% latex microspheres (Fluoresbrite carboxylate YG 0.5 µm; Polysciences, Warrington, PA) for 6 h at 37°C in an atmosphere of 5% CO2. After this period, the cells were washed and prepared for light microscopy. Cells ingesting more than 10 latex particles were designated as phagocytotic cells.

Analysis of cell-surface molecules by flow cytometry
The expression of cell-surface molecules on DCs was determined by flow cytometry. DCs (5x105) were incubated with anti-rat MHC class II (MRC OX6; Serotec), CD11b/c (MRC OX42; Cedarlane Laboratories, Hornby, Ontario, Canada), CD54 (ICAM-1, IA29; Genzyme Techne, Cambridge, MA), B7.1 (CD80; PharMingen, San Diego, CA), or B7.2 (CD86; PharMingen) for 25 min at 4°C. OX21 (anti-human factor I) was used as a negative control. After incubation, the cells were washed three times with 400 µl Hanks’ balanced salt solution and were centrifuged at 1500 g for 5 min. Next, cells were incubated with phycoerythrin (PE)-conjugated anti-mouse immunoglobulin G (IgG)1 (PharMingen) for 30 min, after which the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rat OX62 for 30 min at 4°C. Cells were washed twice and resuspended for analysis. We counted the number of the positive staining cells of PE after gating on OX62-positive cells. Flow cytometric analysis was performed using FACSort (Becton Dickinson, Mountain View, CA), and dead cells were excluded from the analysis on the basis of performed iodide dye exclusion.

DC labeling with carboxyfluorescein diacetate succinimidyl ester (CFDSE)
CFDSE (Molecular Probes, Eugene, OR) was dissolved in dimethyl sulfoxide to 15.6 mM, and a small aliquot (300 µl) was stored in a cuvette sealed with argon gas at -80°C until the experiments. DCs (1x106) were incubated in CFSE solution (20 µl stock solution diluted with 20 ml RPMI) for 30 min at 37°C. The labeled cells were immediately centrifuged through a cushion of heat-inactivated FCS and washed twice with cold suspension medium. The cells were resuspended in 0.5 ml medium and were used within 30 min.

In situ demonstration of migration of DCs in MLNs
Rats were sedated by intraperitoneal injection of pentobarbital sodium (50 mg/kg). CFSE-labeled DCs were injected into the subserosa of the mesenteric border of the small intestine near the cecum by puncture with a glass microsyringe, avoiding blood vessels, and the abdominal walls were closed. The microsyringe was filled with 1 x 106 DCs in 500 µl medium and injected into tissues for 5 min. At different time points, the abdomen was opened via a midline incision, and DC migration into MLNs was observed with an intravital fluorescence microscope (Diaphot TMD-2S, Nikon, Tokyo, Japan). The intestine and the mesentery were kept warm and moist by continuous superfusion with physiological saline at 37°C. Images were captured with a silicon-intensified target camera (C-2400-08, Hamamatsu Photonics, Shizuoka, Japan) as described previously [29 , 30 ].

To obtain a quantitative determination of the time course of DC transport into MLNs, we counted the DCs in cryostat sections of the MLNs. Briefly, after observation under microscopy, MLNs were removed and fixed in periodate lysine paraformaldehyde solution. Thereafter, they were embedded in optimum cutting temperature compound (Miles, Elkhart, IN) before being frozen in a dry ice-acetone bath. Cryostat sections of 6 µm were transferred to poly-L-lysine-coated slides and air-dried for 1 h at 20°C. After washing in PBS (pH 7.4) containing 1% Triton X-100 for 5 min, sections were incubated in PBS containing 5% normal goat serum. The monoclonal mouse anti-rat OX62 antibody was diluted 1:50 in PBS, layered onto the sections that were incubated overnight at 4°C. Sections were incubated with tetramethylrhodamine isothiocyanate-conjugated rabbit anti-mouse Ig for 1 h at room temperature. Sections were rinsed with PBS containing 1% BSA between each step. A coverslip was applied with glycerol jelly. The sections were observed under a fluorescent microscope (ECLIPSE E600, Nikon). The OX62 and CFSE double-positive cells in the MLNs were quantified using an image analyzer and expressed as the number of positively stained cells per field of LNs. For determination, at least five sections were made from one animal, and at least 15 fields of views were randomly counted in each section.

Administration of antibodies and desensitization of DC chemokine receptors
DCs were incubated with monoclonal antibodies (mAb) that functionally block adhesion molecules, ICAM-1 (IA29), or CD11b/c (MRC OX42). As a control, mouse IgGa was used under the same conditions. Cells (1x107) were incubated with 100 µg/ml mAb for 30 min before injection into the cecum.

Recombinant human 6Ckine (CCL21; Genzyme Techne), human stromal cell-derived factor-1{alpha} [CXC chemokine ligand (CXCL)12; R&D Systems], and recombinant rat MIP-3{alpha} (CCL20; Genzyme Techne) were prepared as 0.5 mg/ml stock solutions in saline and were used immediately or were stored as aliquots at -80°C. For desensitization experiments after labeling with CFSE, DCs (1.5x107 cells/ml) were incubated for 45 min with 1 µM chemokine. As Phillips and Ager [31] established that murine or human recombinant forms of CCL21 and CXCL12 could cross-react on rat T lymphocyte receptors and cause their migration in a Transwell assay, we examined the effects of high doses of these chemokines on the DC migration in rats. Controls cells were incubated with saline for 45 min. After washing through serum, the cells were resuspended in 0.5 ml in RPMI and used immediately.

In vitro assay of DC chemotaxis toward chemokines
The chemotactic response was determined in duplicate in multiwell chambers with 5 µm pore size polycarbonate filters (Kurabo Biomedical, Nayagawa, Japan) [32 ]. The cells suspension (106 DCs/0.2 ml) was placed in the upper compartment of the chamber. The lower compartment contained 0.2 ml of a saturating concentration of CCL21, CCL20, or CXCL12. The chamber was incubated in a 5% CO2 incubator for 60 min at 37°C, after which the cells that had migrated into the lower compartment were collected and counted. Results are expressed as the relative number of the cells that migrated across the filter compared with controls (chemotactic number without chemokine in the lower chamber, designated as 1).

Western blot analysis of CCL20 in MLNs
MLNs were homogenized in extraction buffer (100 ml, pH 7.2; Nonidet P-40,0.5 g; Tris-HCl, 0.12 g; NaCl, 0.87 g; NaN3, 0.02 g) containing a mixture of protease inhibitors and were then centrifuged for 10 min at 15,000 rpm at 20,400 g. The protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL) to ensure equal amounts of protein (10 µg) were applied to each lane of the polyacrylamide gel. Samples were prepared for electrophoresis by dilution in 2x sodium dodecyl sulfate (SDS) sample buffer (100 ml, pH 6.8; Tris-HCl, 1.51 g; glycerol, 20 ml; SDS, 4.6 g; p-bromophenacylbromide, 5 mg; dithiothreitol, 100 mM) and were then boiled for 5 min. The soluble supernatants were resolved by SDS-polyacrylamide gel electrophoresis on a 12.5% polyacrylamide gel and were then electrophoretically transferred onto polyvinylidene difluoride membranes (IPVH00010, Millipore, Bedford, MA). The membranes were blocked with Block Ace (UK-B25, Dainippon Pharmaceutical, Osaka, Japan) for 1 h at room temperature. The blots were then incubated with the primary antibody (anti-rat CCL20 mAb, R&D Systems, and anti-actin antibody: A-3937, Sigma-Aldrich Chemical Co.) overnight at 4°C, then washed in PBS-0.05% Tween 20, and further incubated with the secondary antibody [goat F(ab)'2 anti-mouse Ig horseradish peroxidase (HRP) conjugate, Biosource International, Camarillo, CA] for 30 min at room temperature. Bound peroxidase was visualized using a Konica Immunostaining HRP-1000 (Konica, Tokyo, Japan). The membrane was scanned and quantified using Adobe Photoshop elements (Adobe Systems, San Jose, CA).

Analysis of CCL20 and chemokine receptor expression by reverse transcriptase-polymerase chain reaction (RT-PCR)
CCL20 expression in MLNs and CCR6 and CCR7 expression on DCs were determined by RT-PCR. Total RNA was extracted from MLNs or DCs using the RNeasy mini kit (Qiagen, Germany). RT-PCR was then performed using a RNA PCR kit (avian myloblastosis virus), ver.2.1 (Takara Biomedicals, Osaka, Japan), 4 µg RNA, and 0.2 µM primers, according to the manufacturer’s instructions. Total RNA was converted to cDNA using random primers (Invitrogen, Carlsbad, CA). The RT mixture was subjected to PCR with 0.2 µM specific primer in each reaction mixture, which also contained 0.2 mM dNTP, 2.5 U Taq DNA polymerase (Roche Diagnostics), and 2.5 mM MgCl2. The amplification procedure consisted of an initial denaturation at 94°C for 2 min; 40 cycles of denaturation at 50°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 1.5 min; and a final extension at 72°C for 7 min using an iCycler thermal cycler (Bio-Rad Laboratories, Tokyo, Japan). The primers for CCL20 were 5'-TTTGCACCTCCTCAGCCTAAGA-3' and 5'-ACCCCAGCTGTGATCATTTCC-3'. The primers for CCR6 were 5'-CCATGACTGACGTCTACCTATTGAACA-3' and 5'-GAGCAGCTCGAGTCCCATGCCCAGCAG-3'. The primers for CCR7 were 5'-GCTCAACCTGGCCGTGGCAGACATCC-3' and 5'-TTGGATGGCGATCAAGGCCTCC-3'. The primers for ß-actin were 5'-ATGTTTGAGACCTTCAACACC-3' and 5'-TCTCCAGGGAGGAAGAGGAT-3'. The PCR products were resolved on a 12% polyacrylamide gel and were visualized by ethidium bromide staining.

Statistics
All data are expressed as mean ± SEM. Differences among groups were evaluated by one-way ANOVA and Fisher’s post hoc test. Statistical significance was established as P< 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation and phagocytotic function of DCs
Typical DCs were determined as OX62/OX6-positive and HIS24-negative cells (pan B-MHC II+). Giemsa staining showed that most stained cells possessed micropodia. Immunostaining and morphology revealed that the DCs were ~80% pure for intestinal lymph DC and splenic DCs (data not shown). The phagocytotic function of DCs cultured with or without cytokines was examined by their ability to take up latex microbeads. Most of unstimulated DCs from intestinal lymph (86±4%) were latex microbead-phagocytotic, suggesting that a large percentage of these DCs maintains phagocytotic activity. Conversely, the induction of maturation significantly decreased the number of microbead-phagocytotic cells to 13 ± 5% of the unstimulated lymph DCs (P<0.05). The phagocytotic ability of splenic DCs was also lower, specifically 32 ± 5% of the unstimulated lymph DCs (P<0.05).

Expression of cell-surface molecules by DCs
Figure 1 compares the expression of MHC II, CD11b, CD54 (ICAM-1), CD80, and CD86 on DCs from intestinal lymph and spleen. Freshly isolated, intestinal lymph DCs expressed MHC II, ICAM-1, and CD11b/c molecules, but expression of CD80 and CD86 molecules was low (Fig. 1A) . Maturation induction with cytokines significantly increased the expression of CD80, CD86, and MHC II molecules on the intestinal lymph DCs, and there was no significant change in CD11b/c or ICAM-1 expression (Fig. 1B) . Characteristics of the cell-surface molecules of splenic DCs showed an intermediate pattern of CD80, CD86, and MHC II expression compared with freshly isolated and maturated lymph DCs (Fig. 1C) . These results suggest that in terms of cell-surface markers, splenic DCs have a phenotype intermediate between freshly isolated lymph DCs and matured DCs.



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  		Figure 1. Expression of surface molecules on lymph and splenic DCs. DCs were incubated with anti-rat mAb MHC II (MRC OX6), CD11b/c (MRC OX42), CD54 (ICAM-1, IA29), CD80 (B7.1), or CD86 (B7.2). Next, the cells were incubated with PE-conjugated anti-mouse IgG, followed by FITC-conjugated anti-rat OX62. Flow cytometric analysis was performed using FACSort (Becton Dickinson). We counted the number of the positive-staining cells of PE after gating on OX62-positive cells. Representative data from at least four individual measurements are shown. (A) Surface molecules on freshly isolated, intestinal lymph DCs expressing MHC II, ICAM-1, and CD11b/c molecules, but expression of CD80 and CD86 molecules is low. (B) Intestinal lymph DCs after maturation induction with TNF-{alpha}, IL-4, and GM-CSF. Maturation induction significantly increased the expression of CD80, CD86, and MHC II molecules. (C) Splenic DCs without stimulation. The cell-surface molecules of splenic DCs showed an intermediate pattern of CD80, CD86, and MHC II expression compared with freshly isolated and maturated lymph DCs.

 
In situ demonstration of DC migration to MLNs
Figure 2 shows representative images from intravital microscopy of the distribution of fluorescently labeled, intestinal lymph DCs in the marginal sinus of MLNs after injection into the serosa of the small intestine. As shown in Figure 2A , some labeled DCs began to appear in the periphery of MLNs after 12 h, and the number increased at 24 h (Fig. 2B) . After 48 h, DC appearance in the MLNs reached a maximum, and the accumulation of the labeled DCs was intense and diffused in the MLNs (Fig. 2C) . Thereafter, the number of labeled DCs decreased, almost disappearing from the observation field after 72 h (Fig. 2D) . To determine the number of DCs in MLNs, we used cryostat sections of the isolated MLNs. Figure 3A shows the OX62 and CFSE double-positive cells in cryostat sections of MLNs, and Figure 3B compares the number of positively stained cells among three groups (freshly isolated, intestinal lymph DC; maturation-induced lymph DC; and splenic DC). Freshly isolated DCs significantly and dramatically increased their number 48 h after injection into the intestine. In contrast, matured lymph DC and splenic DC accumulation peaked at 24 h rather than 48 h, suggesting that the transportation of these DCs through intestinal lymph is much faster than for freshly isolated, lymph DCs. However, the total number of DCs counted in the MLNs during 72 h was the same for the three groups (freshly isolated lymph DCs, 14.8±1.6; matured lymph DCs, 15.2±1.9; splenic DCs, 14.6±1.1).



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  		Figure 2. Representative intravital microscopic images of the distribution of CFSE-labeled intestinal lymph DCs in the marginal sinus of MLNs. Isolated, intestinal lymph DCs were injected into the subserosa of the small intestine near the cecum, and images were taken at intervals. (A) After 12 h, some labeled DCs began to appear in the periphery of MLNs. (B) After 24 h, the number increased. (C) After 48 h, DC appearance in the MLNs reached a maximum, and (D) 72 h after injection, the number of labeled DCs decreased.

 


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  		Figure 3. (A) Representative picture of the OX62 and CFSE double-positive cells in cryostat sections of MLNs [12 (a), 24 (b), 48 (c), and 72 (d) h after injection of freshly isolated DCs; original magnification, x400]. (B) Time course of the number on transported DCs to MLNs determined from cryostat sections of the isolated LNs at different time points after injection into intestinal subserosa. The migration kinetics of DCs is compared by three types of DCs (isolated intestinal lymph DCs, maturation-induced lymph DCs, and splenic DCs). The y-axis shows the number of DCs per one high power field (HPF) from six animals. For the determination, at least five sections were made from one animal, and at least 15 fields of views were randomly counted in each section and averaged, and then the results from six animals per group were determined. Maturation of intestinal lymph DCs was induced by TNF-{alpha}, IL-4, and GM-CSF. Values are expressed as the mean ± SEM for six animals. *, P< 0.05, versus unstimulated lymph DCs. Freshly isolated DCs significantly increased their number 48 h after injection. In contrast, matured lymph DC and splenic DC accumulation peaked at 24 h rather than 48 h.

 
Effects of adhesion receptor-blocking antibodies and desensitization of DC chemokine receptors by chemokines
We next examined the role of the cell-surface adhesion molecules ICAM-1 and CD11b/c on DC migration kinetics. As shown in Figure 4 , there was no significant change in the migration kinetics of freshly isolated lymph DCs after blocking with anti-ICAM-1 or anti-CD11b/c as compared with administration with control IgG. These antibodies also did not affect the migration kinetics of mature or splenic DCs (data not shown).



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  		Figure 4. Effect of CD11b/c- and ICAM-1-blocking antibodies on the migration kinetics of isolated, intestinal lymph DCs to MLNs. The number of DCs in MLNs was quantitatively determined from cryostat sections of the isolated LNs at different time points. The y-axis shows the number of DCs per one HPF from six animals. As a control, mouse IgGa was used under the same conditions. Values are expressed as the mean ± SEM for six animals. There was no significant change in the migration kinetics after blocking with anti-ICAM-1 or anti-CD11b/c as compared with administration with control IgG.

 
In contrast, our in vivo studies revealed that the several chemokines and their receptors play a significant role in the migration of DCs from intestine toward the MLNs (Fig. 5 ). As shown in Figure 5A , the number of maturation-induced DCs transported to the MLNs was significantly suppressed by prior desensitization with the chemokine CCL21. Thus, CCR7 desensitization by CCL21 induced a disappearance of the peak accumulation at 24 h that had been observed in case of nondesensitized, matured DCs (as shown in Fig. 3 ). Similarly, after blocking of the chemokine receptor CCR7, transportation of splenic DCs to MLNs was also inhibited at 24 h. Conversely, it should be noted that the migration of freshly isolated lymph DCs to MLNs was not affected by CCR7 desensitization, suggesting that the CCL21/CCR7 combination works in the migration process only when the final maturation occurs (data not shown). Instead, the migration of freshly isolated, immature lymph DCs was significantly inhibited by desensitization of the receptor CCR6 with a high dose of CCL20 but not by desensitization of CXC chemokine receptor (CXCR)4 with CXCL12 (Fig. 5B) . Desensitization of CCR6 with CCL20 clearly attenuated the peak accumulation of freshly isolated lymph DCs at 48 h, although it did not completely abolish the peak accumulation at 48 h.



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  		Figure 5. Effect of chemokine receptor desensitization on DC migration kinetics toward MLNs. The number of DCs in MLNs was quantitatively determined from cryostat sections of the isolated LNs at different time points. The y-axis shows the number of DCs per one HPF from six animals. (A) Desensitization of the CCR7 receptor on DCs with CCL21. Maturation-induced DCs from intestinal lymph and splenic DCs are examined. Maturation of intestinal lymph DCs was induced by TNF-{alpha}, IL-4, and GM-CSF. Values are expressed as mean ± SEM for six animals. CCR7 desensitization by CCL21 suppressed the transportation of mature DCs to the MLNs and thus induced a disappearance of the peak accumulation at 24 h that had been observed as shown in Figure 3 . Similarly, after blocking of the chemokine receptor CCR7, transportation of splenic DCs to MLNs was also inhibited at 24 h. (B) Desensitization of CCR6 and CXCR4 receptors of intestinal lymph DCs using CCL20 and CXCL12, respectively. Values are expressed as mean ± SEM for six animals. *, P < 0.05, versus untreated, freshly isolated lymph DCs. The migration of freshly isolated, immature lymph DCs clearly shows peak accumulation at 48 h, but this is significantly inhibited by desensitization of the receptor CCR6 with CCL20, although it did not completely abolish the peak accumulation at 48 h. Desensitization of CXCR4 with CXCL12 did not affect the migration.

 
In vitro chemotactic assay of DCs toward chemokines
Chemotaxis toward CCL21 for the three DC populations (freshly isolated, intestinal lymph DC, matured lymph DC, and splenic DC) was compared in vitro. As shown in Figure 6A , mature DCs from intestinal lymph showed a dose-dependent ability to move toward CCL21, but this ability disappeared after desensitization of CCR7 on mature DCs (data not shown). Splenic DCs showed a similar but slightly weaker chemotaxis to CCL21 than mature lymph DCs (Fig. 6A) . Conversely, freshly isolated, immature DCs from intestinal lymph did not respond to CCL21 (Fig. 6A) . Instead, freshly isolated lymph DCs showed a dose-dependent migration toward CCL20. Conversely, no significant response was induced by CXCL12 (Fig. 6B) . Finally, chemotaxis of immature DCs toward CCL20 was significantly attenuated by desensitization of its receptor, CCR6 (data not shown).



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  		Figure 6. In vitro chemotaxis of DCs toward chemokines. The chemotactic response was determined in multiwell chambers with 5-µm pore size polycarbonate filters. The cell suspension (106 DCs/0.2 ml) was placed in the upper compartment of the chamber. The lower compartment contained 0.2 ml of a saturating concentration of CCL21, CCL20, or CXCL12. The chamber was incubated in a 5% CO2 incubator for 60 min at 37°C, after which the cells that had migrated into the lower compartment were collected and counted. Values are expressed as mean ± SEM (n=6). *, P < 0.05, versus no chemokine treatment (controls). Results are expressed as a relative number of the cells that migrated across the filter compared with controls. (Chemotactic number without chemokine in the lower chamber is designated as 1.) (A) Chemotaxis toward CCL21 for lymph DCs and splenic DCs. Freshly isolated, immature DCs from intestinal lymph did not respond to CCL21. Conversely, mature DCs from intestinal lymph and splenic DCs showed a dose-dependent ability to move toward CCL21. (B) Chemotaxis toward various concentrations of CCL20 and CXCL12 by freshly isolated, intestinal lymph DCs. Freshly isolated lymph DCs showed a dose-dependent migration toward CCL20, but no significant response was induced by CXCL12.

 
CCL20 expression in MLNs and CCR6/7 expression on DCs
To address the role of the CCL20/CCR6 system in immature DC migration into MLNs, we investigated whether MLNs express CCL20 and whether freshly isolated lymph DCs express CCR6. Western blot analysis and RT-PCR analysis demonstrated that CCL20 was expressed in normal MLN tissues at the protein or mRNA level (Fig. 7A and 7B ). Although the expression of CCR6, a receptor for CCL20, was detected in freshly isolated DCs and cytokine-stimulated DCs at the mRNA level using RT-PCR, a stronger expression was observed in freshly isolated DCs compared with that in maturation-induced DCs (Fig. 7C) . We also detected CCR7 mRNA expression in freshly isolated DCs and cytokine-stimulated DCs, but in this case, we found a stronger expression in maturation-induced DCs (Fig. 7D) .



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  		Figure 7. (A) Expression of CCL20 mRNA on MLNs determined by RT-PCR. MLNs expressed a 101-bp CCL20 mRNA. RT(-) shows a control without RT. (B) Western blot analysis of CCL20 protein expression in MLNs. CCL20 expression can be detected at 31 kDa under normal conditions. As a positive control, recombinant rat CCL20 (540-RM-025; R&D Systems) is used. (C) Expression of CCR6 mRNA on freshly isolated, intestinal lymph DCs (Immature) and cytokine-stimulated, matured DCs (Mature) determined by RT-PCR. Freshly isolated DCs and maturation-induced DCs expressed a 425-bp CCR6 mRNA, but freshly isolated DCs showed a stronger expression than that of mature DCs. (D) Expression of CCR7 mRNA on freshly isolated, intestinal lymph DCs (Immature) and cytokine-stimulated, mature DCs (Mature) determined by RT-PCR. Freshly isolated DCs and maturation-induced DCs expressed a 382-bp CCR7 mRNA, with a stronger expression in mature DCs. These show a representative picture from three experiments with similar results.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Investigations of DC biology have predominantly used in vitro-generated and ex vivo-isolated DCs from mice and humans. It is, however, often difficult to relate these results to the behavior of DCs in vivo. The rat model we have used in the current studies permits analysis of DCs in a nearly physiological setting. Although it has been suggested that DCs migrate from the periphery only under conditions of overt stimulation [11 ], our results confirm the previous hypothesis that lymph-borne DCs represent a population of cells constitutively and continuously migrating from the intestine into MLNs in the absence of any overt stimulation [13 ]. The migratory kinetics of freshly isolated, intestinal lymph DCs agrees with a previous report that rat DCs spend an average of 3 days in the intestinal wall before migrating to the draining nodes [11 ]. Thus, the turnover of DCs in rat gut is more rapid than nonmucosal sites such as skin epidermis or solid organs, where the DC turnover time has been estimated at 2–4 weeks [33 ].

Older DCs are continually migrating from the periphery and are replaced by immature precursors [13 ]. These cells are always present in afferent lymph that access the T cell areas but not in efferent lymph, indicating that most of the migrating DCs die after their arrival in lymphoid tissue [10 ]. Therefore, DCs in MLNs could be considered as end-stage DCs [13 , 34 ]. However, in the present study, we showed that DCs in intestinal lymph are not significantly matured, as assessed by their ability to take up fluorescent particles and their low expression of costimulatory molecules. This suggests that a final maturation signal may be received only when DCs enter the microenvironment of the MLNs [13 , 34 ]. Recently Geissmann et al. [35] showed that Langerhans cells and/or their dermal precursors accumulate within LNs in an immature state during chronic, inflammatory disease, and they suggest that Langerhans cell maturation and migration might occur as separable events and that maturation need not progress strictly upstream of migration [35 , 36 ]. If immature DCs promote naive T cells to develop a tolerogenic phenotype [37 ], one might speculate that immature DCs also traffic efficiently to LNs from the periphery. Huang et al. [38 ] recently reported that a discrete subpopulation of DCs isolated from intestinal lymph was specialized for the transport of apoptotic cells from intestinal to draining LNs. These immature, lymph-borne DCs could be involved in tolerance rather than immunity by helping T cells become tolerant to self-antigens derived from apoptotic cells [39 ].

Our results also revealed that if lymph-borne DCs were induced to a final state of maturation by cytokines, their migration toward MLNs was accelerated, suggesting that once DC maturation programs are launched in the intestine by inflammatory conditions, it could largely promote DC migration through intestinal lymph. In other words, if DC maturation is induced, they will exhibit a faster migration toward MLNs. However, even if maturation accelerated the DC velocity to reach the MLNs, maturation per se did not significantly increase the total number of transported DCs. It is also intriguing that CD11c+, {alpha}E-integrin+, MHC class II+ DCs are found in spleen and that they are probably at a more advanced stage of maturation than freshly collected lymph DCs. Our study further demonstrated that the migration kinetics of splenic DCs was similar to that of maturation-induced, intestinal DCs, supporting the possibility that the maturation state of DCs mainly regulates the speed of their migration into the MLNs.

What kinds of molecules on DCs modulate the movement to the MLNs? Although CD11b [40 ] or ICAM-1 [41 ] has been shown to mediate DC adhesion to vascular endothelial cells and despite the fact that we found that ICAM-1 plays a functional role in the lymphocyte exit from Peyer’s patches to intestinal lymph [42 ], neither CD11b/c nor ICAM-1 was involved in the migration of DCs through intestinal lymph. Instead, our experimental data clearly showed that the combination of certain chemokines and their ligands plays a pivotal role in the migration kinetics of DCs toward the MLNs. Namely, if CCL21 is involved, faster migration is induced. Conversely, if CCL20 is involved, slower migration occurs. It has been reported that although immature DCs respond to many CC and CXC chemokines, mature DCs lose their responsiveness to most of these chemokines and instead, acquire responsiveness to CCL19 and CCL21, which are specifically expressed in the T cell-rich areas where mature DCs home [21 , 22 ]. Therefore, as observed in CCR7- or CCL21-deficient mice [24 ], CCL19 and CCL21 have a key role in the accumulation of antigen-loaded, mature DCs in T cell-rich areas of the draining LNs. These reports concur with our present results using matured DCs. Surprisingly, though, freshly isolated lymph DCs were unable to respond to CCL21 in spite of their surface expression of MHC II, suggesting that the migration of immature DCs to MLN is CCR7-independent. Instead, our study demonstrates the importance of CCL20/CCR6 in the migration of steady-state, intestinal DCs to the MLNs. Recently, a role for CCL20 and CXCL12 in immature DC recruitment has been suggested in lymphoid neogenesis and ectopic lymphoid organ-like rheumatoid arthritis synovium [43 44 45 ]. However, this is in contrast to the migratory pattern of DCs to other peripheral LNs, where CCR7 is considered to be necessary for migration of all DCs, regardless of maturation state, to afferent lymphatic vessels [36 ]. The relative scarcity of DCs in LNs of CCR7-deficient mice supports this notion, and Martín-Fontecha et al. [46] recently showed that CCR7-/-DCs subcutaneously injected into the mouse foodpad failed to migrate to the draining LNs. Future work should aim to clarify this intriguing difference in chemokine receptor use between mesenteric and peripheral LNs of DC migration.


   ACKNOWLEDGEMENTS
 
This study was supported in part by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture of Japan and by grants from Keio University, School of Medicine, and the National Defense Medical College.

Received June 1, 2003; revised October 31, 2003; accepted November 13, 2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
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 DISCUSSION
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